The present invention relates to methods of ex-vivo expansion of stem cells, such as hematopoietic stem cells, by co-culture with mesenchymal cells, to expanded populations of renewable stem cells expanded by co-culture with mesenchymal cells, and to their uses. In particular, fetal and/or adult hepatic, and umbilical cord blood, bone marrow or peripheral blood derived hematopoietic stem cells expanded ex-vivo along with mesenchymal cells, according to the methods of the present invention, can be transplanted into recipients, for example, following myeloablation.
Expansion of Stem and Progenitor Cell Populations:
While many methods for stimulating proliferation of stem and progenitor cell populations have been disclosed [see, for example, Czyz et al, Biol Chem 2003; 384:1391-409; Kraus et al., (U.S. Pat. No. 6,338,942, issued Jan. 15, 2002); Rodgers et al. (U.S. Pat. No. 6,335,195 issued Jan. 1, 2002); Emerson et al. (Emerson et al., U.S. Pat. No. 6,326,198, issued Dec. 4, 2001) and Hu et al. (WO 00/73421 published Dec. 7, 2000) and Hariri et al (US Patent Application No. 20030235909)] few provide for reliable, long-term expansion, without the accompanying differentiation that naturally occurs with growth of stem or progenitor cells in culture.
Up until recently, expansion of renewable stem cells has been achieved either by growing the stem cells over a feeder layer of fibroblast cells, or by growing the cells in the presence of the early acting cytokines thrombopoietin (TPO), interleukin-6 (IL-6), an FLT-3 ligand and stem cell factor (SCF) (Madlambayan G J et al. (2001) J Hematother Stem Cell Res 10: 481, Punzel M et al. (1999) Leukemia 13: 92, and Lange W et al. (1996) Leukemia 10: 943). While expanding stem cells over a fibroblast feeder layer results in vast, substantially endless cell expansion, expanding stem cells without a feeder layer, in the presence of the early acting cytokines listed above, results in an elevated degree of differentiation (see Leslie N R et al. (Blood (1998) 92: 4798), Petzer A L et al. (1996) J Exp Med Jun 183: 2551, Kawa Y et al. (2000) Pigment Cell Res 8: 73).
Recently, however, methods for feeder-layer free expansion of stem cells ex-vivo have been disclosed. PCT IL99/00444 to Peled et al., filed Aug. 17, 1999, which is incorporated by reference as if fully set forth by reference herein, and from which the present invention derives priority, disclosed methods of imposing proliferation yet restricting differentiation of stem and progenitor cells by treating the cells with chelators of transitional metals. While reducing the invention to practice, they uncovered that heavy metal chelators having a high affinity for copper, such as tetraethylpentamine (TEPA), greatly enhanced the fraction of CD34+ cell and their long-term clonability in cord-blood-derived, bone marrow-derived, and peripheral blood derived stem and progenitor cells. Facilitation of proliferation while inhibiting differentiation was also observed in erythroid progenitor cells, cultured mouse erythroleukemia cells, embryonal stem cells, and hepatocytes in primary hepatocyte culture treated with TEPA.
PCT IL03/00062, also to Peled et al., filed Jan. 23, 2003, which is incorporated by reference as if fully set forth herein, and from which the present invention derives priority, discloses a similar effective promotion of long term ex vivo stem cell proliferation, while inhibiting differentiation, using TEPA-Cu chelates as well as the chelator TEPA. Surprisingly, this effect of TEPA and TEPA-chelates was also demonstrated using as a starting population an un-selected peripheral mononuclear fraction. The results described therein clearly show that stem and progenitor hematopoietic cells may be substantially expanded ex vivo, continuously over at least 12 weeks period, in a culture of mixed (mononuclear fraction) blood cells, with no prior purification of CD34+ cells.
PCT IL 03/00064, also to Peled et al., filed Jan. 26, 2003, which is incorporated by reference as if fully set forth herein, and from which the present invention derives priority, teaches the ex-vivo expansion and inhibition of hematopoietic stem and progenitor cells using conditions and various molecules that interfere with CD38 expression and/or activity and/or with intracellular copper content, for inducing the ex-vivo expansion of hematopoietic stem cell populations. The small molecules and methods include linear polyamine chelators and their chelates, nicotinamide, a nicotinamide analog, a nicotinamide or a nicotinamide analog derivative or a nicotinamide or a nicotinamide analog metabolite, a PI 3-kinase inhibitor, conditions for reducing a capacity of the hematopoietic mononuclear cells in responding to retinoic acid, retinoids and/or Vitamin D and reducing the capacity of the cell in responding to signaling pathways involving PI 3-kinase.
Surprisingly, the inventors also showed that exposure of hepatocytes in primary culture to the small molecules, and conditions described hereinabove stimulated hepatocyte proliferation, greatly expanding the fraction of undifferentiated and immature hepatocytes (as determined by α-feto-protein expression, OC3 marker expression and oval cell morphology). Thus, using the methods described, adult stem and progenitor cells of hematopoietic and non-hematopoietic origin can provide expanded populations of cells for transplantation into endodermally derived organs.
PCT IL 03/00681, also to Peled et al, filed Aug. 17, 2003, which is incorporated by reference as if fully set forth herein, and from which the present invention derives priority, discloses methods of ex-vivo expanding a population of hematopoietic stem cells present, even as a minor fraction, in hematopoietic mononuclear cells, without first enriching the stem cells, while at the same time, substantially inhibiting differentiation of the hematopoietic stem cells. Cells thus expanded can be used to efficiently provide ex-vivo expanded populations of hematopoietic stem cells without prior enrichment of the hematopoietic mononuclear cells for stem cells suitable for hematopoietic cell transplantation, for genetic manipulations for cellular gene therapy, as well as in additional application such as, but not limited to, adoptive immunotherapy, implantation of stem cells in an in vivo cis-differentiation and trans-differentiation settings, as well as, ex-vivo tissue engineering in cis-differentiation and trans-differentiation settings.
PCT IL 2004/000215, also to Peled et al., filed Mar. 4, 2004, which is incorporated by reference as if fully set forth herein, and from which the present invention derives priority, further demonstrated the self-renewal of stem/early progenitor cells, resulting in expansion and inhibition of differentiation in stem cells of hematopoietic origin and non-hematopoietic origin by exposure to low molecular weight inhibitors of PI 3-kinase, disruption of the cells' PI 3-K signaling pathways.
PCT IL 2005/000753, also to Peled et al., filed Jul. 14, 2005, which is incorporated by reference as if fully set forth herein, and from which the present invention derives priority, discloses methods of expansion and inhibition of differentiation in stem cells of hematopoietic origin by exposure to inhibitors of the sirtuin family of enzymes, such as nicotinamide and splitomycin, resulting in extensive expansion of a cell population that displays phenotypic and functional characteristics of primitive hematopoietic progenitor cells.
PCT IL 2004/000644, also to Peled et al., filed Jul. 15, 2004, which is incorporated by reference as if fully set forth herein, and from which the present invention derives priority, discloses methods of ex-vivo expansion of endodermally-derived and non-endodermally-derived progenitor and stem cells, expanded populations of renewable progenitor and stem cells and their use for transplantation into solid organs for repopulation and treatment of diseases of endodermally derived organs such as liver and pancreas.
Hematopoietic Cell—Mesenchymal Cell Interactions:
Mature blood cells are derived from undifferentiated stem and progenitor cells in a highly complex series of maturational and divisional steps that occur in different tissues during embryonic development. The microenvironment seems to be an important factor influencing the proliferative activity and differentiation process of the stem and progenitor cells by local positive and negative signaling to the target cells (Williams D A. Stem cell model of hematopoiesis. In: Hoffman R, et al., eds. Hematology: Basic Principles and Practice. 2nd ed. New York, N.Y.: Churchill Livingstone; 1995:180). Within the bone marrow stroma there exists a subset of nonhematopoietic cells referred to as mesenchymal stem (MSC) or mesenchymal progenitor cells (MPC). Mesenchymal progenitor cells do not express the typical hematopoietic antigens, CD45, CD34, HLA-DR and CD14 and they are positive for CD105, CD49b, CD73 and HLA class 1. However, none of these markers is specific for mesenchymal progenitor cells. Recently, Thomas et al (US Patent Application No. 20040058397) has disclosed additional combinations of antibody markers to identify and enrich mesenchymal progenitor cells.
The following 3 mechanisms have been proposed to explain the role of non-hematopoietic stromal cells in the regulation of proliferation and differentiation of hematopoietic stem cells (Long M W. Exp Hematol. 1992; 20:288-301): (a) cytokine receptor-ligand interaction, (b) interaction between adhesion molecules on hematopoietic cells and stromal cells, or with components of the extracellular matrix, or (c) direct cell-to-cell communication between stromal cells or between stromal cells and hematopoietic cells.
Gap Junctions: Very little is known about the regulatory mechanisms of direct cell-to-cell communication in the hematopoietic microenvironment. Intercellular gap junctions represent the most well known intercellular communication system, and they are characterized by the existence of plaques of narrow channels between contacting cells. Each channel is formed by two hemichannels or hemiconnexons, and each one of them is contributed by one of two adjacent cells. A hemiconnexon is an oligomeric assembly of 6 polypeptide subunits, or connexins. Different tissue-specific connexins have been characterized and cloned (Kumar N M, Gilula N B Cell, 1996; 84:381-388). These channels form the only known system for direct diffusional exchange of ions and small molecules (ie, molecular weight<1000) between contacting cells (Spray D C. Circ Res. 1998; 83:679-681). Nutrients and second messengers can be quickly transported in this way through cell communication networks.
There are few reports analyzing the gap junctions in hematopoietic tissues. Rosendaal et al (J Cell Sci. 1994; 107:29-37) reported on the basis of immunohistological studies that, in adult mouse bone marrow, the connexin (Cx43) gap junction epitopes are rare, but are up-regulated 80- to 100-fold in the marrow of the neonate or after forced stem cell division (by administering 5-fluorouracil [5-FU] or irradiation). This up-regulation occurs soon after an insult, before recognizable blood cells form, and around the time at which primitive stem cells are triggered to go into cycle, suggesting the presence of a latent network of gap junctions in normal hematopoietic tissues. Significantly, it has been reported that global blockade of all gap junctions and intercellular communication by amphotericin B reversibly inhibits the cobblestone area (CA) formation and hematopoiesis in stroma-containing cultures (Rosendaal M, et al. Leukemia. 1997; 11:1281-1289). In osteoblasts (Steinberg T H, et al. EMBO J. 1994; 13:744-750) and in fetal fibroblasts (Martyn K D, et al., Cell Growth Differ. 1997; 8:1015-1027), it has been shown that different gap junction proteins create channels with different conductance properties, suggesting that the gap junctions' contribution to regulation of cell functions might also differ between the different gap junctions. Thus, stromal-hematopoietic cell interaction may be mediated by gap junctions and/or other cell-to-cell connections.
Mesenchymal cells can be cultured. Two bone-marrow culture systems introduced in the mid-1970's have evolved as favored media for the in vitro analysis of mesengenesis and hematopoiesis. The Friedenstein culture system is based on the isolation of nonhematopoietic cells through their tendency to adhere to plastic. Once isolated, a monolayer of homogeneous, undifferentiated stromal cells is then grown in the culture medium, in the absence of hematopoietic cells. The stromal cells from this system have the potential to differentiate into discrete mesenchymal tissues, namely bone, cartilage, adipose tissue and/or muscle depending on specific growth supplements (Friedenstein, et al, Exp Hematol 1976 4, 267-74). In 1977, Dexter, et al. developed another bone marrow culture system for the study of hematopoiesis. (Dexter et al. J Cell Physiol 1977, 91:335-44). The Dexter culture does not require isolation of the mesenchymal cells before culturing, thus the monolayer of stromal cells is grown in the presence of hematopoietic cells. Greenberger later modified the Dexter system by the addition of hydrocortisone to the culture medium, making it more reproducible (Greenberger, Nature 1978 275, 752-4).
Co-culture of mesenchymal and hematopoietic cells has been previously reported. U.S. Pat. No. 6,030,836 to Thiede et al discloses the use of bone marrow mesenchymal stem cells or adipocytes in co-culture with autologous bone marrow hematopoietic stem cells, and the enhanced survival and expansion of hematopoietic progenitors such as CD34+, CD 34+/90+ and CD34+/CD14+. However, Thiede et al report that while the hematopoietic stem cell fraction of the cultured cells is not depleted, it grows only marginally.
Experiments with mesenchymal stem cells have indicated that they are “invisible” to the immune system. Normally, co-culturing cells from different individuals (allogeneic cells) results in T cell proliferation, manifested as a mixed lymphocyte reaction (MLR). However, when human mesenchymal stem cells are contacted with allogeneic T lymphocytes, in vitro, they do not generate an immune response by the T cells, i.e., the T cells do not proliferate, indicating that T cells are not responsive to mismatched mesenchymal stem cells (Maitra, et al., Bone Marrow Transplant 2004, 33:597-604), despite the fact that the human mesenchymal stem cells express all of the class I and class II MHC surface molecules that render them immunogenic. Mesenchymal stem cells also actively reduce the allogeneic T cell response in mixed lymphocyte reactions in a dose dependent manner, and mesenchymal stem cells from different donors do not exhibit specificity of reduced response with regard to MHC type.
Thus, the use of mesenchymal cells, and mesenchymal cells culture in transplantation has been investigated. Using NOD/SCID mice, Noort, et al have demonstrated that co-transplantation of mesenchymal cells isolated as non-hematopoietic cells from fetal lung CD34+ cells significantly enhanced the engraftment of hematopoietic stem cells (Noort et al Exp Hematol 2002; 30:870-78). Similarly, Maitra et al (Maitra, et al., Bone Marrow Transplant 2004, 33:597-604) have demonstrated the successful repopulation of NOD/SCID mice with limited numbers of hematopoietic stem cells, augmented by co-infusion with unrelated human mesenchymal stem cells. Significantly, no enhancement was observed with a co-infusion with mouse mesenchymal stem cells. Human mesenchymal stem cells culture has also been shown to support the ex-vivo propagation of CD34+ cells, in the absence of direct contact between the mesenchymal and hematopoietic cells in culture, and enhance transplantation. (Sumner, et al, Cytother 2001 3; 422a). Therapeutic use of mesenchymal stem cells and stem cell culture has been investigated. Administration of expanded mesenchymal stem cell cultures has been proposed for treatment of articular disorders (US Patent Application No. 20040151703 to Ha, et al), Hurler syndrome and metachromatic leukodystrophy (Koc, et al, Bone Marrow Transplant. 2002; 30:215-22) and for connective tissue engraftment, as well as hematopoietic cell engraftment (U.S. Pat. No. 6,355,239, to Bruder et al). Koc, et al (J Clin Oncol, 2000, 18:307-16) reported the co-infusion of culture expanded autologous bone marrow mesenchymal stem cells (in DMEM and bovine fetal serum medium) and peripheral blood progenitor cells in 32 breast cancer patients after high dose chemotherapy, with no observed toxicity or reduced engraftment related to the mesenchymal cell administration. Infusion of allogeneic bone marrow derived mesenchymal stem cells for Hurler syndrome and metachromatic leukodystrophy (Koc, et al, Bone Marrow Transplant. 2002; 30:215-22) also indicated no toxicity and possible therapeutic value. Recently, Lazarus, et al (Biol. Blood Marrow Transplant. 2005; 11:389-98) reported the co-administration of culture expanded mesenchymal stem cells and hematopoietic stem cells from sib-matched donors to 46 hematopological malignancy patients following high dose chemotherapy, with no toxicity and an increased probability of successful transplant in the co-administered group.
Thiede et al (U.S. Pat. No. 6,030,836) disclose the use of co-cultured bone marrow hematopoietic stem cells and mesenchymal stem cells (or adipocytes), enriching the CD34+, CD34+/90+ and CD34+/CD14+ fraction of hematopoietic stem cells for engraftment. McIntosh et al (U.S. Pat. Nos. 6,368,636 and 6,875,430) teach the reduction of immune response to cellular transplant, and reduction in graft versus host disease, by infusion of mesenchymal stem cells before, after, or along with the transplanted cells. Seshi, et al (US Patent Application No. 2003000308) teaches the isolation of mesenchymal progenitor cells expressing different multiple cellular differentiation markers, such as fat, osteoblasts, smooth muscle and fibroblast markers, for treatment of graft versus host disease, and for enhanced transplantation.
Based on the above descriptions, it is clear that there is thus a widely recognized need for, and it would be highly advantageous to have, methods enabling efficient ex-vivo expansion of hematopoietic stem cells using co-culture of hematopoietic stem cells with mesenchymal cells, yielding large numbers of stem cell populations for transplantation, alone or along with the mesenchymal cells.